Geranyl diphosphate synthase (GPP synthase) is one of a family of enzymes called prenyltransferases that catalyze C.sub.5 elongation reactions to form the linear (acyclic) precursors of the various terpenoid families. GPP synthase catalyzes the condensation of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) to form geranyl diphosphate (GPP) which is the immediate, C.sub.10 acyclic precursor of the monoterpenes (Wise, M. L. and Croteau, R., in Cane, D. E., ed., "Comprehensive Natural Products Chemistry: Isoprenoids, Vol. 2", Elsevier Science, Oxford, 1999. ps. 97-154) (FIG. 1). Farnesyl diphosphate synthase (FPP synthase), a related prenyltransferase, utilizes GPP and IPP as substrates to form farnesyl diphosphate (FPP), which is the immediate, C.sub.15 precursor of the sesquiterpenes (FIG. 1). Another prenyltransferase, geranylgeranyl diphosphate synthase (GGPP synthase), catalyzes the condensation of farnesyl diphosphate (or DMAPP) and IPP to form geranylgeranyl diphosphate (GGPP) which is the immediate C.sub.20 precursor of the diterpene family (FIG. 1). Other types of prenyltransferases can utilize FPP, GGPP and IPP as substrates to form very long chain molecules, such as natural rubber. Poulter C. D. and Rilling, H. C., Accts. Chem. Res. 11: 307-313 (1978); Scolnik, P. A. and Bartley, G., Plant Mol Biol. Rep. 14: 305, 307 (1996); Ogura, K. and Koyama, T., Chem. Rev. 98: 1263-1276 (1998).
The basic reaction mechanism for all of these prenyltransferases is the same, and consists of three steps (see FIG. 2 in which the reaction catalyzed by geranyl diphosphate synthase is presented as illustrative of the general reaction mechanism). With reference to FIG. 2, in the first step an allylic diphosphate ester (2a) is ionized to the stable carbonium ion (2b). The carbonium ion then attacks the double bond of isopentenyl diphosphate (2c) to yield another carbonium ion (2d). In the final step of the cycle, a proton is eliminated from the newly formed carbonium ion (2d) to form a terpenoid containing a new allylic double bond (2e). In the reaction catalyzed by GPP synthase, the allylic diphosphate ester is dimethyl allyl diphosphate (FIG. 1 and FIG. 2). In the reactions catalyzed by FPP synthase and GGPP synthase the allylic diphosphate ester is geranyl diphosphate and farnesyl diphosphate, respectively (FIG. 1).
Unlike FPP synthase and GGPP synthase, which produce GPP as an intermediate and which are nearly ubiquitous (Ogura, K. and Koyama, T., in Ogura, K. and Sankawa, U., eds., "Dynamic Aspects of Natural Products Chemistry" Kodansha/Harwood Academic Publishers, Tokyo, pp. 1-23, 1997), geranyl diphosphate synthase is largely restricted to plant species that produce abundant quantities of monoterpenes. Because both FPP synthase and GGPP synthase produce only negligible levels of GPP as a free intermediate on route to FPP and GGPP ((Ogura, K. and Koyama, T., in Ogura, K. and Sankawa, U., eds., "Dynamic Aspects of Natural Products Chemistry" Kodansha/Harwood Academic Publishers, Tokyo, pp. 1-23, 1997)), it is geranyl diphosphate synthase that provides the crucial link between primary metabolism and monoterpene biosynthesis and that serves as the essential driver of monoterpene biosynthesis (Wise, M. L. and Croteau, R., supra).
Any attempt, therefore, to exploit recombinant methods to increase the yield of monoterpene-producing (essential oil) species, or to genetically engineer the monoterpene biosynthetic pathway into any non-producing species (e.g., field crops, fruit-bearing plant species and animals) requires access to a geranyl diphosphate synthase gene or cDNA clone. Co-expression of geranyl diphosphate synthase along with a selected monoterpene synthase, such as (-)-limonene synthase (Colby et al., J Biol. Chem. 268:23016-23024, 1993), and any subsequent pathway enzymes, should allow production of the corresponding monoterpene product(s) from simple carbon substrates, such as glucose, in any living organism.
Monoterpenes are utilized as flavoring agents in food products, and as scents in perfumes (Arctander, S., in Perfume and Flavor Materials of Natural Origin, Arctander Publications, Elizabeth, N.J.; Bedoukian, P. Z. in Perfumery and Flavoring Materials, 4th edition, Allured Publications, Wheaton, Ill., 1995; Allured, S., in Flavor and Fragrance Materials, Allured Publications, Wheaton, Ill., 1997. Monoterpenes are also used as intermediates in various industrial processes. Dawson, F. A., in The Amazing Terpenes, Naval Stores Rev., Mar./Apr., 6-12, 1994. Monoterpenes are also implicated in the natural defense systems of plants against pests and pathogens. Francke, W. in Muller, P. M. and Lamparsky, D., eds., Perfumes: Art, Science and Technology, Elsevier Applied Science, NY, N.Y., 61-99, 1991; Harborne, J. B., in Harborne, J. B. and Tomas-Barberan, F. A., eds., Ecological Chemistry and Biochemistry of Plant Terpenoids, Clarendon Press, Oxford, 399-426, 1991; Gershenzon, J and Croteau, R in Rosenthal, G. A. and Berenbaum, M. R., eds., Herbivores: Their Interactions with Secondary Plant Metabolites, Academic Press, San Diego, 168-220, 1991.
There is also substantial evidence that monoterpenes are effective in the prevention and treatment of cancer (Elson, C. E. and Yu, S. G., J Nutr. 124: 607-614, 1994.). Thus, for example, limonene, perrilyl alcohol and geraniol have each been shown to have chemotherapeutic activity against a very broad range of mammalian cancers (see, for example, (1) limonene, Elegbede et al., Carcinogenesis 5:661-665, 1984; Elson et al., Carcinogenesis 9:331-332, 1988; Maltzman et al., Carcinogenesis 10:781-785, 1989; Wattenberg, L. W. and Coccia, J. B., Carcinogenesis 12:115-117, 1991; Wattenberg, L. W. and Coccia, J. B., Carcinogenesis 12:115-117, 1991; Haag et al., Cancer Res. 52:4021-4026, 1992; Crowell, P. L. and Gould, M. N., CRC Crit. Rev. Oncogenesis 5:1-22, 1994; (2) perillyl alcohol, Mills et al., Cancer Res. 55:979-983, 1995; Haag, J. D. and Gould, M. N., Cancer Chemother. Pharmacol. 34:477-483, 1994; Stark et al., Cancer Lett. 96:15-21, 1995 and (3) geraniol, Shoff et al., Cancer Res. 51:37-42, 1991; Yu et al., J. Nutr. 125:2763-2767, 1995; Burke et al., Lipids 32:151-156, 1997.).
Cancer cells can be modified to produce therapeutic amounts of a monoterpene having anti-cancer properties by targeting the cognate monoterpene synthase protein to cancer cells, or by introducing a monoterpene synthase gene into cancer cells. This approach to cancer therapy is complicated, however, by the fact that the natural distribution of geranyl diphosphate synthase is largely restricted to plant species that produce abundant quantities of monoterpenes. Thus, animal cells do not naturally produce the monoterpene precursor geranyl diphosphate. Consequently, the genetic manipulation of cancer cells to produce endogenous monoterpenes having anti-cancer properties requires the introduction of a gene encoding geranyl diphosphate synthase, together with a gene encoding a monoterpene synthase that produces a monoterpene having anti-cancer properties. Similarly, if the protein targeting approach is utilized, both geranyl diphosphate synthase protein and monoterpene synthase protein must be targeted to cancer cells.
Standard protein targeting techniques can be used to introduce geranyl diphosphate synthase along with a monoterpene synthase, such as limonene synthase (Colby et al., J. Biol. Chem. 268:23016-23024, 1993), into animal cells with specific targeting to tumors. See, e.g., Wearley, L. L., Critical Reviews in Therapeutic Drug Carrier Systems, 8(4): 331-394, 1991; Sheldon, K et al., Proc. Nat'l. Acad. Sci. USA., 92(6): 2056-2060, 1995. In addition, standard gene therapy techniques can be used to target a GPP synthase gene and a monoterpene synthase gene to cancerous cells for endogenous synthesis of monoterpenes having anti-cancer properties. For reviews of gene targeting technology see; Mahato R. I et al., Pharmaceutical Research 14(7): 853-859, 1997; Rosenthal, F. M. and Mertelsmann, R., Onkologie 20(1): 26-34, 1997; Buckel, P., Trends in Pharmacological Sciences 17(12): 450-456, 1996; Roth, J. A. and Cristiano, R. J., J. Nat'l Cancer Inst. 89(1): 21-39, 1997; Ledley, F. D, Pharmaceutical Research 13(11): 1595-1614, 1996.
To date, extracts containing geranyl diphosphate synthase activity have been isolated from several plant sources, including grape (Clastre et al., Plant Physiol. 102:205-211, 1993); geranium (Suga, T. and Endo, T., Phytochemistry 30:1757-1761, 1991); sage (Croteau, R. and Purkett, P. T., Arch. Biochem. Biophys. 271:524-535, 1989) and Lithospermum (Heide, L. and Berger, U., Arch Biochem. Biophys. 273:331-338, 1989). Only the enzyme from grape has been purified to homogeneity (Clastre et al., supra). The structures and properties of prenyltransferase enzymes and genes are reviewed in, K. Ogura and T. Koyama, Chem. Rev. 98:1263-1276 (1998), and in T. Koyama and K. Ogura, "Isopentenyl Diphosphate Isomerase and Prenyltransferases," in Cane, D.E., ed., Comprehensive Natural Products Chemistry: Isoprenoids, Vol. 2, Elsevier Science, Oxford, 1999, pp. 69-96.
U.S. patent Ser. No. 5,876,964 and copending, international patent application PCT/US98/21772, each disclose the isolation and sequence of cDNA molecules encoding what was initially characterized as a geranyl diphosphate synthase protein. The putative geranyl diphosphate synthase protein exhibited only a small fraction of the biological activity of native geranyl diphosphate synthase extracted from plant tissue. In view of the disclosure of the present patent application, the putative geranyl diphosphate synthase protein disclosed in U.S. patent Ser. No. 5,876,964, and in copending, international patent application PCT/US98/21772, is now known to be the small subunit of geranyl diphosphate synthase which exists, in its native, fully functional form, as a heterodimer including a small subunit and a large subunit. The present patent application discloses the isolation and sequence of a Mentha cDNA encoding geranyl diphosphate synthase large subunit, and enables the isolation of additional nucleic acid molecules encoding geranyl diphosphate synthase large subunit.